Download Brood pheromone suppresses physiology of extreme longevity in

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The Journal of Experimental Biology 212, 3795-3801
Published by The Company of Biologists 2009
doi:10.1242/jeb.035063
Brood pheromone suppresses physiology of extreme longevity in honeybees
(Apis mellifera)
B. Smedal1, M. Brynem2, C. D. Kreibich1 and G. V. Amdam1,3,*
1
Department of Chemistry, Biotechnology and Food Science, University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway,
2
Department of Animal and Aquacultural Sciences, University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway and
3
School of Life Science, Arizona State University, Tempe, P.O. Box 874501, AZ 85287, USA.
*Author for correspondence ([email protected])
Accepted 19 September 2009
SUMMARY
Honeybee (Apis mellifera) society is characterized by a helper caste of essentially sterile female bees called workers. Workers
show striking changes in lifespan that correlate with changes in colony demography. When rearing sibling sisters (brood),
workers survive for 3–6 weeks. When brood rearing declines, worker lifespan is 20 weeks or longer. Insects can survive
unfavorable periods on endogenous stores of protein and lipid. The glyco-lipoprotein vitellogenin extends worker bee lifespan by
functioning in free radical defense, immunity and behavioral control. Workers use vitellogenin in brood food synthesis, and the
metabolic cost of brood rearing (nurse load) may consume vitellogenin stores and reduce worker longevity. Yet, in addition to
consuming resources, brood secretes a primer pheromone that affects worker physiology and behavior. Odors and odor
perception can influence invertebrate longevity but it is unknown whether brood pheromone modulates vitellogenin stores and
survival. We address this question with a 2-factorial experiment where 12 colonies are exposed to combinations of absence vs
presence of brood and brood pheromone. Over an age-course of 24 days, we monitor the amount of vitellogenin stored in
workers’ fat body (adipose tissue). Thereafter, we track colony survival for 200 days. We demonstrate that brood rearing reduces
worker vitellogenin stores and colony long-term survival. Yet also, we establish that the effects can result solely from exposure
to brood pheromone. These findings indicate that molecular systems of extreme lifespan regulation are integrated with the
sensory system of honeybees to respond to variation in a primer pheromone secreted from larvae.
Supplementary material available online at http://jeb.biologists.org/cgi/content/full/212/23/3795/DC1
Key words: vitellogenin, fat body, nurse load, lifespan regulation, colony survival.
INTRODUCTION
Feral honeybees live in a wide range of climates, from tropical Africa
to the temperate zone of Northern Europe (Ruttner, 1988). A colony
usually consists of one reproductive queen, some hundred drones
(males) and many thousand sister workers that perform a variety of
tasks, including cleaning, building comb, nursing brood and foraging
for nectar and pollen (Winston, 1987). In temperate climates, the
lifespan distribution of worker bees is strongly bimodal (Fukuda
and Sekiguchi, 1966; Fluri and Imdorf, 1989). During favorable
conditions in summer, young workers conduct tasks inside the nest
such as nursing, and 2–3 weeks later they initiate foraging (Seeley,
1982). The majority of bees die within 1–2 weeks of their first
foraging flight (Neukirch, 1982; Visscher and Dukas, 1997) with a
resulting adult lifespan of 3–6 weeks for summer workers. However,
when the favorable season ends, brood rearing and foraging ceases.
Instead of dividing labor between nest tasks and foraging activities,
the workers enter the diutinus ‘winter bee’ stage (Amdam and
Omholt, 2002), and can survive for 20 weeks or longer (Maurizio,
1950).
The bimodal distribution of worker bee lifespan (with peaks
around 3–6 weeks vs 20 weeks or longer) can be an adaptation to
a strongly seasonal environment; during favorable conditions,
summer bees sustain colony growth and reproduction by raising
drones, sister workers and virgin queens and by taking part in
swarming whereas diutinus workers allow the society to endure cold
winter months when resources are limited (Omholt, 1988).
Determining how adult bees can enter the diutinus stage is important,
not only for understanding the bee’s biology of survival but for
improving our general understanding of aging processes (Muench
et al., 2008). Honeybees have received increasing attention in aging
research (Brandt et al., 2005; Remolina and Hughes, 2008) because
some aspects of their lifespan regulation (Amdam and Omholt, 2002;
Guidugli et al., 2005; Seehuus et al., 2006; Nelson et al., 2007) can
differ from the molecular signaling systems of senescence studied,
e.g. in Caenorhabditis elegans (reviewed by Kenyon, 2005) and
Drosophila melanogaster (reviewed by Toivonen and Partridge,
2009). Many studies have addressed how diutinus bees develop.
Cessation of flight activity, decreasing daylength and falling
temperatures may play a role (Kefuss and Nye, 1970; Huang and
Robinson, 1995) but the vast majority of research has focused on
how reduced brood rearing influences worker physiology (Maurizio,
1950; Haydak, 1963; Free and Racey, 1968; Avitabile, 1978;
Winston, 1980; Omholt, 1988).
The extended lifespan of diutinus bees correlates with an
increased amount of stored lipids and proteins in their hemolymph
(blood) and fat body [analogous to liver and adipose tissue (Koehler,
1921; Maurizio, 1954; Fluri et al., 1977; Shehata et al., 1981; Fluri
et al., 1982)]. Fat body nutrient content correlates with survival
capability in insects (Haunerland and Shirk, 1995; Hahn and
Denlinger, 2007). Honeybee fat body also influences worker lifespan
by acting as the synthesis organ of vitellogenin, a glyco-lipoprotein
(Seehuus et al., 2006). Vitellogenin is a female-specific yolk
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3796 B. Smedal and others
precursor in many oviparous species that has taken on new functions
in honeybees. The protein is utilized in brood food production
(Amdam et al., 2003) and in the regulation of foraging behavior. It
can also extend worker lifespan independent of behavior (Nelson
et al., 2007), possibly by scavenging free radicals (Seehuus et al.,
2006) and enhancing innate immunity (Amdam et al., 2004b;
Amdam et al., 2005a).
In the absence of brood, vitellogenin accumulates in the
hemolymph of workers, and this accumulation characterizes diutinus
bees (Amdam et al., 2004a; Amdam et al., 2005b). We hypothesized
that the diutinus bees’ physiology of extreme longevity could
develop because less vitellogenin was lost to the production of brood
food (Amdam and Omholt, 2002; Amdam et al., 2009). This
proposition builds on previous work by Omholt (Omholt, 1988),
who suggested that the nurse load of young honeybee workers affects
longevity: a low nurse load can increase lifespan whereas a high
nurse load has the opposite effect. In addition to consuming colony
resources, however, larval brood secrete a primer brood pheromone,
a blend of 10 fatty acid methyl and ethyl esters (Le Conte et al.,
1994) produced by the salivary glands (Le Conte et al., 2006). This
pheromone affects worker brain gene expression (Alaux et al., 2009),
gland physiology (Pankiw et al., 2008) and behavior (Pankiw, 2004).
Odors and odor perception influence longevity in C. elegans
(Alcedo and Kenyon, 2004) and D. melanogaster (Libert et al., 2007)
but the effect of brood pheromone on worker bee survival is
unknown.
As brood rearing and nurse load decline in colonies toward the
end of summer, so does the amount of brood pheromone. Previous
work that explains diutinus bee development as a function of brood
rearing does not fully account for this fact. To resolve how the
amount of brood in honeybee colonies can affect worker lifespan,
it is necessary to decouple the effects of nurse load and brood
pheromone. To achieve this, we used a 2-factorial design that took
into account that workers are exposed to nurse load and pheromone
from larvae when brood is present, while they encounter neither
when brood is absent. Workers were exposed to brood pheromone
in the absence of brood by using an established synthetic pheromone
blend that consists of the same mixture of 10 fatty acid methyl and
ethyl esters as is secreted by fourth–fifth instar larvae (Le Conte et
al., 2001). We measured stored vitellogenin in biopsies from fat
body of 3–4-, 7–8- and 23–24-day-old workers, and we monitored
the long-term survival of colonies. Our data confirm that brood
rearing reduces the amount of stored vitellogenin in fat body and
the long-term survival of colonies. Yet, we also demonstrate that
brood pheromone interacts with brood rearing and worker age to
influence vitellogenin, and that the decline in vitellogenin content
and colony survivorship is achieved by exposure to brood
pheromone alone. These results establish a new understanding of
how brood rearing regulates honeybee lifespan.
MATERIALS AND METHODS
Our factorial experiment was composed of four treatments that
corresponded to all possible combinations of presence vs absence
of larval brood (factor‘brood’) and synthetic brood pheromone
(factor‘BP’). We replicated the design three times at colony level,
using 12 honeybee colonies in total (Fig.1).
Honeybees
In August 2007, 12 colonies with about 3000 worker bees each (Apis
mellifera carnica L.) were prepared in six 2-compartment hive
bodies at the apiary of the University of Life Sciences, Aas, Norway.
Every hive box was divided in the middle by an excluder wall (wood)
Colony 1
Colony 2
Colony 3
Colony 4
Colony 5
Colony 6
Colony 7
Colony 8
Colony 9
Colony 10
Colony 11
Colony 12
Fig.1. The 2-factorial design that represented all possible combinations of
the presence vs absence of larval brood (‘brood’) and synthetic brood
pheromone blend (‘BP’), resulting in four experimental treatments. Each
treatment combination was applied to an entire honeybee colony, and the
design was replicated three times. The resulting 12 colonies were
maintained in 2-compartment hive boxes. Treatments were quasirandomized between boxes so the same set of treatments never cooccurred more than once between colonies in a single hive box, as
indicated by color codes: white‘brood’ and ‘BP’ absent; blue‘brood’
present, ‘BP’ absent; gray‘brood’ absent, ‘BP’ present; blue/gray upward
diagonal‘brood’ and ‘BP’ present. Black bars on either side of each hive
unit indicate the entrances of the colonies.
to form the two equally sized compartments that each accommodated
one colony (Fig.1). The colonies were prepared as described before
(Amdam et al., 2004a). Briefly, to ensure that the distribution of
queen pheromone (central to colony integrity) (Winston, 1987) and
brood could be carefully controlled during the experiment, each
colony was given one queen that was caged on a wax comb in the
center of the nest. The 12 queens were sisters, freely mated at the
same certified A. m. carnica mating station. By caging, the queens’
motility and egg-laying behavior were constrained so that colonies
would not produce their own brood. In parallel, all colonies received
synthetic queen pheromone for the duration of the experiment
(following the manufacturer’s instructions, BeeBoost, Pherotech
International, Delta, BC, Canada) (Amdam et al., 2004a).
Three colonies were assigned to each of the four treatments from
the 2-factorial experimental design (Fig.1). Overall, the four
factorial treatments were quasi-randomized between hive boxes, so
that the same set of treatments never co-occurred more than once
between two colonies in a single hive body (Fig.1). Presence of
brood was achieved by adding two combs with eggs and young
larvae to colonies every fifth day of the experiment (egg-to-larval
development is three days, while larval-to-pupal development is six
days). Each set of brood comb replaced the previous set, which
ensured the continuous presence of open brood and prevented brood
from emerging as adults in the colonies. Through the duration of
the experiment, we made an effort to match the relative treatment
amount of larval brood to the relative treatment amount of synthetic
brood pheromone (below) by using a similar number of larval
equivalents (LEqu) per colony (exposure to about 1000 larvae daily).
Colonies assigned absence of brood treatment received sham
handling. The presence of synthetic pheromone blend was achieved
by treating colonies with an established mix of 10 fatty acid methyl
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brood pheromone and honeybee lifespan
3797
and ethyl esters (Le Conte et al., 1994). For application, small glass
dispensers were filled with 250ml sugar syrup (Bifor®, Danisco,
Denmark) and the brood pheromone was mixed in by careful stirring.
For honeybee colonies, experimental delivery of pheromones is
routinely done by feeding (e.g. Le Conte et al., 2001; Leoncini et
al., 2004). All colonies received one dispenser of syrup freshly mixed
each day at 10:00h. Colonies assigned absence of brood pheromone
treatment received dispensers with sugar syrup only.
As soon as this experimental setup was established, each colony
received 700 newly emerged (<24h old) adult worker bees (A. m.
carnica) that were marked with paint on the thorax to convey host
colony identity. Each group of 700 bees consisted of an equal mix
of workers from five different colony sources. This mixture ensured
an equal and broad genotypic distribution of marked bees among
all the colonies of the experiment. The hives were monitored daily
to ensure that the caged queens were alive, that the treatment scheme
was applied consistently and that colonies were similar in strength
(number of workers) and remained healthy (without symptoms of
known honeybee pathogens or disease).
Bees were sampled until day 24 of the experiment and the four
factorial treatments were continued for two more weeks. Thereafter,
treatments were discontinued by releasing the queens from the cages,
and by removing brood and brood pheromone blend. The 12 colonies
were wintered in a controlled environment room at 0°C and 50–60%
RH (relative humidity). Over the next three weeks, colonies were
observed every fifth day to verify that worker mortality declined,
a pattern consistent with successful overwintering (Fluri and Imdorf,
1989; Mattila et al., 2001). This basic validation was facilitated by
replacing the standard bottom-boards of the hives with a custommade set that had built-in drawers. As workers died, they fell from
the colony cluster into the drawers, and could be counted. After the
three weeks, we inspected the colonies one final time to confirm
that the queens were alive and that all hive units were healthy (see
above). The colonies were scored as alive or dead after 200 days.
factorial design, five independent sections (biopsies) were collected
from the fat bodies of each of 15 individuals, which were selected
randomly from the three replicate colonies that received each
treatment (summing to a total of 60 bees). Biopsies were from ages
3–4, 7–8 and 23–24 days. The five separate biopsies from each of
the 60 workers were processed independently during five separate
and unique replicate rounds of immunostaining to ensure that
inference from the material overall was not confounded by the
potentially great intra-individual heterogeneity of insect fat body
tissue (Jensen and Borgesen, 2000) and by potential errors linked
to technical replication in immunofluorescence analysis.
For each staining round, tissue sections were dried onto
SuperFrost®Plus slides, rinsed with PBS-T (phosphate buffer saltsolution pH7.2, with 0.02% Triton X100) 3⫻5min, washed
3⫻5min with PBS-T and blocked with 2% BSA (Sigma-Aldrich,
Steinheim, Germany) in PBS-T for 60min at room temperature. After
3⫻15min washes with PBS-T, sections were incubated overnight
at 4°C with a polyclonal (rabbit) anti-vitellogenin antibody at 1:500
(raised against 180kDa honeybee vitellogenin, Pacific Immunology,
Ramona, CA, USA); specificity was tested and confirmed previously
(Seehuus et al., 2007). The negative control was incubated with PBST, as verified before (Seehuus et al., 2007). After 3⫻5min washes
in PBS-T, the samples were incubated with a polyclonal anti-rabbit
antibody coupled to the fluorochrome Cy3 (AffiniPure Goat AntiRabbit IgG, Jackson ImmunoResearch Europe, Newmarket, Suffolk,
UK), dilution 1:200, overnight at 4°C. For negative control staining,
sections were incubated with the secondary antibody only (Seehuus
et al., 2007). Finally, samples were washed 3⫻5min in PBS-T and
subsequently mounted in 50% glycerol/PBS. Images were acquired
with a confocal laser scanning microscope (Leica TCS SP5, Leica
Microsystems, Wetzlar, Germany). In total, this design of sample
processing resulted in 300 images.
Synthetic brood pheromone blend
A scoring key was developed by choosing images that represented
incremental differences in signal intensity. Increments were
discerned as ascending from the least to the most intense positive
staining among the 300 images. This approach identified seven
images that were assigned a relative gray-scale intensity from 1 (least
intense image) to 7 (most intense image). The resulting scoring key
(supplementary material Fig.S1) was evaluated and approved by
two additional subjects that did not take part in the subsequent
scoring of the sample material. Four independent observers without
prior expectations of image intensities were asked to score the
intensity of the remaining 293 images on a scale from 1 to 7 based
on the scoring key. Observers were blind to the treatment identity
of the images. After scoring, one consensus value was calculated
for each image as the mean of the four independent scores that it
had received.
A factorial analysis of variance (ANOVA) model was used to
analyze the resulting dataset of 300 image intensities. ‘brood’, ‘BP’
(each with two levels) and ‘age’ (three levels) were coded as fixed
main effects. ‘Staining round’ (five replicate staining series on
biopsies from each of 60 workers) was coded as a random factor.
The dataset adhered to the assumptions of ANOVA, after the criteria
of normality (estimated by a normal probability plot of residuals
from the analysis) and homogeneity of variances (determined by
Levene’s test). Post-hoc comparisons were made with Fisher’s LSD
test.
The data on colony long-term survival were analyzed with
factorial ANOVA using a one-sided test criterion (see Results).
This blend was as described before (Le Conte et al., 1994), i.e.
methyl palmitate 5%, methyl oleate 18%, methyl stearate 8.5%,
methyl linoleate 6%, methyl linolenate 10.5%, ethyl palmitate 7.5%,
ethyl oleate 21%, ethyl stearate 11%, ethyl linoleate 2% and ethyl
linolenate 10% (Fluka, Buchs, Switzerland). Exposure to brood
pheromone was estimated in LEqu according to Le Conte et al. (Le
Conte et al., 1994). Each treated colony of about 3700 bees received
1000 LEqu daily, corresponding to about 1/4–1/3 LEqubee–1day–1.
After the blend was prepared, it was aliquoted into microcentrifuge
tubes and stored at –20°C until applied (described above).
Sampling of worker bees and tissue fixation
Starting on day 3 and ending on day 24 of the experiment, marked
worker bees were sampled from all colonies. Bees were taken
quickly from the apiary to the laboratory and dissected on ice. The
sting apparatus and gut were removed before the abdomen was
stored in PP-test tubes (Greiner Bio-One, Monroe, NC, USA), fixed
in formaldehyde and embedded in London Resin White (Electron
Microscopy Science), as described before (Seehuus et al., 2007), to
provide resin blocks with embedded abdominal tissue ready for
sectioning.
Immunofluorescence
Semi-thin sections (1–2m) of resin-embedded material were cut
with a diamond knife using a Reichert Jung ultra-microtome
(Ontario, Canada). For each of the four treatment groups of the
Semi-quantitative scoring of fat body vitellogenin storage and
statistic analysis
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3798 B. Smedal and others
‘Brood’ and ‘BP’ were fixed main effects and the dependent
variable, survivorship, was coded as 0 (dead) or 1 (alive). ANOVA
is not an optimal model for such categorical outcome variables but
the approach can accommodate factorial designs and is widely in
use (Jaeger, 2008). One colony was excluded because it did not
winter (see above) (Fluri and Imdorf, 1989; Mattila et al., 2001).
All statistics used Statistica 6.0 (StatSoft, Tulsa, OK, USA).
As an independent factor in ANOVA, ‘BP’ did not explain
variation in vitellogenin storage (F1.42, P0.23). Vitellogenin
accumulation, however, was significantly affected by ‘BP’ in
interaction with ‘brood’ (F13.73, P<0.0005) and with ‘age’
(F7.52, P<0.001), implying that ‘BP’s influence on physiology
was conditional on the social environment (i.e. if brood was present
or not) and worker ontogeny. Explicitly, in fat body from 3–4-dayold workers, exposure to brood pheromone tended to increase the
amount of vitellogenin (Fisher’s LSD test, P<0.05). By contrast,
the presence of brood pheromone strongly reduced stored
vitellogenin in biopsies taken from 23–24-day-old bees (Fisher’s
LSD test, P<0.0001). This level of suppression was indistinguishable
from the negative influence brood rearing had on the amount of
granules with positive immunostaining in biopsies from the same
age group (Fisher’s LSD test, P0.11). In fact, as long as 23–24day-old workers had been exposed to brood pheromone, the mean
amount of vitellogenin stored in their fat bodies was the same
regardless of whether larval brood was present or absent in colonies
(Fisher’s LSD test, P0.88) (Fig.2D–F). In line with this manysided interplay between treatment factors and worker age, the dataset
confirmed a significant three-factor interaction between ‘brood’,
‘BP’ and ‘age’ (F7.55, P<0.001).
Summing the data, our experimental design successfully
decoupled effects of nurse load and brood pheromone as the fat
body’s physiology developed with worker age. The experiment
showed that a repression of stored vitellogenin occurs when brood
RESULTS
Vitellogenin stored in individual fat bodies
Stored vitellogenin protein was detected as immunostained granules
(Seehuus et al., 2007) in anterior tissue-sections (biopsies) of
workers’ abdominal fat body (Fig.2A–C). We found that the factors
‘brood’ and ‘age’ significantly influenced the mean amount of
vitellogenin stored in the fat body (F19.75, P<0.0001 and F77.33,
P<0.0001, respectively; d.f.238). The presence of brood in colonies
reduced the amount of vitellogenin detected in biopsies from 7–8and 23–24-day-old bees (Fisher’s LSD test, P<0.001), while
increasing worker age led to a greater amount of stored protein: fat
body tissue from 23–24-day-old workers contained more
vitellogenin on average than fat body tissue collected from younger
bees (Fisher’s LSD test, P<0.0005) (Fig.2D–F). This time-course
of vitellogenin increase is in line with previous data on how the
hemolymph vitellogenin titer can develop with worker age (Amdam
et al., 2004a; Amdam et al., 2005b), particularly during autumn (Fluri
et al., 1982).
3–4 days
7–8 days
A
23–24 days
B
C
BP
1
0
0
1
0
1
0
1
Brood
7
D
E
F
BP
BP
Mean amount of stored vitellogenin
6
5
0
1
Fig.2. Semi-quantitative levels of vitellogenin stored in fat body
(adipose tissue). The amount of vitellogenin was scored in 300
sections (biopsies) taken from the anterior abdominal fat bodies
of 60 worker honeybees. (A–C) Image micrographs of fat body
cells immunostained for vitellogenin (white), as obtained from
3–4-, 7–8- and 23–24-day-old bees. Each image represents the
mean score given to its assigned treatment group. The four
treatment groups of the experiment are identified by the
factorial combinations of ‘0’ and ‘1’ indicators below and to the
left of each panel. Zero represents the absence of brood or
synthetic brood pheromone blend, and 1 indicates presence.
Scale bar50m. (D–F). Factorial bar graphs (d.f.238) of
corresponding mean + 95% confidence interval, showing the
semi-quantitative amount of vitellogenin stored in the samplegroups exemplified by panels A–C. For each time-point (3–4,
7–8 and 23–24 days), panels D–F show the relative quantities
of stored vitellogenin achieved in the absence vs presence of
brood (assigned as 0 and 1 on the x-axes), and the absence
and presence of synthetic pheromone blend (assigned as blue
vs gray bars, respectively). Trend lines connect corresponding
means.
4
3
2
1
0
0
1
0
1
0
1
Brood
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Brood pheromone and honeybee lifespan
is present, which represents a combined influence of nurse load and
brood pheromone. Yet, the experiment also revealed that a similar
repression is achieved with brood pheromone alone. The greatest
mean amount of stored vitellogenin, accordingly, characterized the
fat bodies of 23–24-day-old workers from colonies treated with
neither brood nor brood pheromone (Fig.2F), conditions that
replicate the colony setting where diutinus bees develop (Maurizio,
1950; Fluri and Imdorf, 1989).
Long-term survival of colonies
The 12 colonies were wintered in a controlled environment room
and scored as alive or dead after 200 days. Two hundred days is an
extended but not uncommon duration of diutinus bee lifespan
(reviewed by Amdam and Omholt, 2002). From our knowledge on
vitellogenin stored in fat bodies (see above), we expected a priori
that pre-wintering exposure to brood or brood pheromone could only
reduce the survival probability of colonies. Therefore, the dataset
was analyzed with factorial ANOVA using a one-sided test criterion,
which assumes that the null hypothesis is rejected only by outcomes
located entirely at one end of the probability distribution
(Montgomery, 1997). We found that ‘brood’ (F3.81, P<0.05) and
‘BP’ (F3.81, P<0.05) influenced colony survival (d.f.8). As
predicted, the ability to survive 200 days was reduced after prewintering exposure to brood or brood pheromone (Fig.3). Colonies
treated with both factors, furthermore, were significantly less likely
to survive compared with colonies exposed to neither brood nor
synthetic brood pheromone blend (Fisher’s LSD test, P<0.03). This
latter group also tended to outlive colonies that were exposed to
either brood or synthetic pheromone before overwintering (Fisher’s
LSD test, P0.05). The survival probability of the latter two
treatment groups (either brood or synthetic blend) was identical
(Fisher’s LSD test, P0.22).
DISCUSSION
Mean survival probability
Our data provide new insights into mechanisms that allow worker
bees to develop extreme longevity potential. We show that changes
in storage protein physiology, which can affect worker survival
directly (Koehler, 1921; Maurizio, 1950; Maurizio, 1954;
Haunerland, 1996; Seehuus et al., 2006), are influenced by a primer
BP
0
BP
1
1
0
0
1
Brood
Fig.3. Factorial bar graphs (d.f.8) for colony long-term survival. After 200
days, colony survivorship was scored as 0 (dead) or 1 (alive). The resulting
mean + 95% confidence interval for colony survival probability is graphed
for each treatment group. The absence vs presence of brood is assigned
on the x-axis as 0 and 1, respectively. The absence and presence of
synthetic pheromone blend is each indicated by blue vs gray bars,
respectively.
3799
pheromone secreted by larval brood. This finding calls for a revised
understanding of how diutinus bees develop and of how successful
overwintering is achieved by honeybee colonies.
Exposure to synthetic brood pheromone blend tended to increase
the amount of vitellogenin in the fat bodies from young 3–4-dayold bees. This result may be understood in light of a recent study
by Pankiw and coworkers (Pankiw et al., 2008), who showed that
brood-rearing colonies treated with synthetic brood pheromone blend
consumed more pollen substitute. Pollen is the major protein source
of honeybees (Winston, 1987). In parallel with the increased
consumption of pollen substitute by colonies, Pankiw and coworkers
found that the hypopharyngeal head glands of nest workers had a
higher protein content. Hypopharyngeal glands are active in brood
food synthesis (reviewed by Crailsheim, 1990). Pankiw et al.
(Pankiw et al., 2008) hypothesized that brood pheromone stimulates
young workers to consume more pollen to ensure a brood-foodproduction-capacity that is ramped up to balance the perceived
demand. Increased consumption of pollen by young bees also
correlates with increased hemolymph levels of vitellogenin (Bitondi
and Simões, 1996). Thus, we propose that the greater amount of
vitellogenin granules seen after 3–4 days of synthetic brood
pheromone treatment (Fig.2A,D) results from increased production
of vitellogenin enabled by a higher consumption of pollen.
In 23–24-day-old workers, our treatment scheme revealed a
significant interaction between factors ‘brood’ and ‘BP’ on the
amount of vitellogenin stored in fat body. Brood suppressed
vitellogenin stores in the absence of synthetic pheromone blend,
and synthetic pheromone blend suppressed vitellogenin stores in
the absence of brood; yet, there was no further reduction (no additive
effect) seen in fat bodies from workers that were exposed to both
factors (Fig.2C,F). The lack of an additive effect of brood and
synthetic pheromone blend was apparent also in biopsies from 3–4day-olds (no further increase in storage in the presence of both
factors) (Fig.2D) and 7–8-day day-olds (no further decrease in the
presence of both factors) (Fig.2E). After the 23–24 days, thus, the
same mean vitellogenin stores were achieved regardless of whether
worker bees were exposed to brood treatment, brood pheromone or
both. How is this pattern explained? The brood treatment of our
experiment consisted of larvae that secreted brood pheromone. A
primer pheromone treatment constituent, therefore, was shared
between ‘brood’ and ‘BP’. Because exposure to either factor had
the same physiological outcome after 23–24 days, we hypothesize
that brood pheromone is the causal element that suppresses further
vitellogenin storage as workers grow older. The lack of an additive
effect in the presence of both factors, regardless of worker age, could
be due to sensory adaptation (Wark et al., 2007) in the insects’ doseresponse to pheromone (Marion-Poll and Tobin, 1992).
The positive effect of synthetic brood pheromone on
vitellogenin stores in 3–4-day-old bees and the negative effect in
later life (as suggested in 7–8-day-olds and significant after 23–24
days) (Fig.2E,F) indicate that brood pheromone has a dual
function. Brood pheromone may increase pollen consumption in
workers (Pankiw et al., 2008), enhancing their capacity to produce
brood food and to store a surplus from vitellogenin synthesis.
But, the pheromone may also inhibit workers from developing a
physiology of extensive vitellogenin storage, ensuring that more
vitellogenin remains free in hemolymph (Amdam and Omholt,
2002) and ready to be converted into brood food (Amdam et al.,
2003). This effect on the stored amount of protein would impact
the young workers (our 3–4-day-olds) less than the more mature
worker bees (our 7–8- and 23–24-day-olds), because the
cumulative amount of vitellogenin that potentially can be stored
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3800 B. Smedal and others
increases with time (Amdam and Omholt, 2002). These putative
functions of the brood pheromone are in line with recent data
from Fischer and Grozinger, who proposed that honeybee queen
mandibular primer pheromone can modify nutrient storage
pathways in worker fat body (Fischer and Grozinger, 2008).
We found that pre-wintering exposure to brood and brood
pheromone decreased the long-term survival probability of the
experimental colonies (Fig.3). In contrast to the data from tissues,
the colony-level results point to an additive effect of the treatment
scheme, in that colonies kept in the presence of both brood and
synthetic pheromone blend did less well in our survival test (all
died before the completion of the experiment). The negative
influence of brood rearing was indistinguishable from the negative
effect of synthetic brood pheromone blend. This outcome supports
our hypothesis that worker longevity potential, which translates
into colony-level survival capability (Amdam and Omholt, 2002),
is influenced by primer brood pheromone rather than the nurse
load placed on workers. It has already been proposed that brood
rearing shortens worker life and can lead to colony deaths in
winter (Eischen et al., 1984; Omholt, 1988; Fluri and Imdorf,
1989; Amdam and Omholt, 2002) but the cause–effect relationship
was previously explained by the metabolic costs of caregiving
(Amdam et al., 2009).
Our colony-level experiment revealed an additive effect of the
treatment combinations on survival but a similar summation was
absent for the vitellogenin storage physiology of the fat body. How
does this pattern emerge? As noted already, the treatment scheme
was extended for two weeks after we collected the final set of
histological samples (see Materials and methods). Furthermore, our
study focused on a single predictor of survival, fat body vitellogenin
stores, while other physiological variables that influence worker
survival might also have been affected by the treatments. These
factors include the amount of stored lipid and carbohydrate (Koehler,
1921; Shehata et al., 1981; Toth and Robinson, 2005). Thus, the
work presented here does not exclude that our factorial treatment
scheme had additive effects on worker physiology. We speculate
that we observed the colony-level summation of these latent and
many-sided traits of workers indirectly – through the outcome of
our survival test.
Taken together, our data show for the first time that exposure to
brood pheromone is sufficient to explain variation in fat body
vitellogenin stores that may predict age at death in honeybee
workers. This effect of pheromone alone is supported by data on
the long-term survival of colonies. Our results revise the current
knowledge base on how diutinus workers develop in temperate
climates, and therefore, through our work, survival patterns of
honeybees can be better understood. Adding to this, our findings
provide evidence for how a primer pheromone from young siblings
can act on older sisters to shorten lifespan. This relationship should
be of interest to research centered on the interface between sensory
systems and aging regulation (Alcedo and Kenyon, 2004; Libert et
al., 2007), which currently is poorly understood.
ABBREVIATIONS
BP
LEqu
LSD
brood pheromone (factorial treatment factor)
larval equivalents
least significant difference
We thank Y. Le Conte for helpful advice, O. Gjermundrød for assistance with
colonies, and C. Brent, O. Rueppell and K. Traynor for comments on the
manuscript. Support to G.V.A. was provided by the Research Council of Norway
(#175413, 180504, 185306 and 191699), the U.S. National Science Foundation
(#0615502), and the PEW Foundation.
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